feasibility study of in vitro drug sensitivity assay of ...out of the 120 patients only 14 met the...
TRANSCRIPT
1Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505
To cite: Papp E, Steib A, Abdelwahab EMM, et al. Feasibility study of in vitro drug sensitivity assay of advanced non- small cell lung adenocarcinomas. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505
► Additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ bmjresp- 2019- 000505).
Received 6 October 2019Revised 9 May 2020Accepted 12 May 2020
For numbered affiliations see end of article.
Correspondence toDr Judit E Pongracz; pongracz. e. judit@ pte. hu
Feasibility study of in vitro drug sensitivity assay of advanced non- small cell lung adenocarcinomas
Emoke Papp,1 Anita Steib,2 Elhusseiny MM Abdelwahab,3,4 Judit Meggyes- Rapp,2,3 Laszlo Jakab,5 Gabor Smuk,6 Erzsebet Schlegl,7 Judit Moldvay,7,8 Veronika Sárosi,1 Judit E Pongracz 3,4
Lung cancer
© Author(s) (or their employer(s)) 2020. Re- use permitted under CC BY- NC. No commercial re- use. See rights and permissions. Published by BMJ.
AbstrActBackground Despite improved screening techniques, diagnosis of lung cancer is often late and its prognosis is poor. In the present study, in vitro chemosensitivity of solid tumours and pleural effusions of lung adenocarcinomas were analysed and compared with clinical drug response.Methods Tumour cells were isolated from resected solid tumours or pleural effusions, and cryopreserved. Three- dimensional (3D) tissue aggregate cultures were set up when the oncoteam reached therapy decision for individual patients. The aggregates were then treated with the selected drug or drug combination and in vitro chemosensitivity was tested individually measuring ATP levels. The clinical response to therapy was assessed by standard clinical evaluation over an 18 months period.Results Based on the data, the in vitro chemosensitivity test results correlate well with clinical treatment response.Conclusions Such tests if implemented into the clinical decision making process might allow the selection of an even more individualised chemotherapy protocol which could lead to better therapy response.
IntroductIonWhile the recently improved treatment strat-egies have resulted in better survival statistics in many cancers, in non- small cell lung cancer (NSCLC) the 1- year overall survival at a locally advanced or metastatic stage barely exceeds 20%.1 Although key mutations aid clinical decision- making and facilitate the applica-tion of targeted therapies, radical improve-ments have not been observed and the 5- year survival rate remains at approximately 5%.1 Although next- generation sequencing has become a cornerstone of therapy guidance,2 clinical decision- making remains difficult due to the histological diversity of NSCLC (adeno, squamosous, large cell) and the variation of the mutation characteristics of the different subtypes.3 Based on clinical guidelines, patients with advanced lung adenocarcinoma (AC) are tested for the presence of Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR)
mutations and rearrangements involving anaplastic lymphoma kinase (ALK).4 Unfortu-nately, analysis of tumour mutations can only describe the mutations existing at the specific location where the sample was taken from and at the time of sample taking. By the time therapy is selected, additional mutations may have occurred.5 6 Due to the diversity of histology as well as mutations in NSCLC, the first- line treat-ment of locally advanced or inoperable cancer is platinum based, which can in itself dramat-ically increase the mutation rate.7 The rela-tively slow acting immunotherapies are only considered as an alternative in specific cases8; therefore, chemotherapy remains the prin-cipal treatment modality in advanced NSCLC. To improve chemotherapy response rates, drug sensitivity assays have been under intense investigation.9 It was recognised that tissues derived from the original tumour represent the tumour composition suitably well to test chem-osensitivity on freshly isolated tumour cells in vitro.10 Most published tests, however, have not been performed on advanced NSCLC.9 While the statistical analysis of data in the current literature involving different tumours looks convincing, clinicians remain wary of such tests due to the clinical complexity of individual treatment responses. In the present study, we have performed in vitro drug sensitivity analysis in advanced NSCLC AC samples, to investigate sensitivity to currently recommended drugs and compared the results to clinical therapy response.
MAterIAls And MethodsResected solid tissue samples were digested using a Miltenyi Tumor Dissociation Kit (Miltenyi Biotec, Auburn, USA). Cells from pleural fluid were centrifuged (600g, 10 min), then isolated by Ficol separation. Red blood cells were removed by Red Blood Cell Lysis Buffer (Roche, Mannheim, Germany). Cells
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from
2 Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505
Open access
Figure 1 Study design. All the samples were freshly cryopreserved as single cell suspensions, then thawed when the oncoteam made a decision for therapy. Both the resected tissue and the tumour- enriched pleural effusion stained positive for TTF1 and both sample types were routinely tested for Kirsten rat sarcoma 2 viral oncogene homolog, epidermal growth factor receptor and anaplastic lymphoma kinase mutations. Single cell suspensions were cryopreserved and stored at −80°C until used. Samples were thawed and placed into three- dimensional (3D) aggregate cultures, then treated with the corresponding chemotherapeutic agent(s) selected by the oncoteam. Patient therapy responses were monitored and compared with the in vitro assay results. PD, progressive disease; SD, stable disease; TFF1, Thyroid transcription factor 1.
were cryopreserved using Cryo- SFM (PromoCell, Heidel-berg, Germany) and stored at −80°C until used.
Cryopreserved tumour cells were combined with normal human lung fibroblast cells (1:1) and then aggre-gated in a low- attachment 96- well plate (Corning, New York, USA).10
Tissue aggregates were treated with chemotherapeutic compounds (Selleckem, Munich, Germany) selected by the oncoteam.
Concentrations for in vitro treatments were based on the literature11 12 and in vitro concentration tests performed in our laboratory. Drug concentrations were as follows: cisplatin (7 µM),13 14 carboplatin (CBP; 100 µM),14 vinorel-bine (150 nM),15 gemcitabine (30 µM),16 paclitaxel (100 nM),17 pemetrexed (10 µM),16 erlotinib (100 nM)18 and gefitinib (100 nM).19 Treatments were carried out for 48 hours at 37°C in 5% CO2 atmosphere in four parallels.
In vitro viability assay was performed using the three- dimensional (3D) CellTiter Glo (Promega, Madison, USA) kit, measured in a PerkinElmer Plate Reader (PerkinElmer, Waltham, USA).
CT, MRI, chest X- ray and abdominal ultrasound methods were used for Response Evaluation Criteria
In Solid Tumours (RECIST V.1.1) evaluations.20 In the selected patient populations, only stable (SD) and progressive (PD) diseases were distinguished. The in vitro chemosensitivity results were compared with the patients’ clinical responses to chemotherapy at 2–3 months and patients were monitored over 18 months. One- way anal-ysis of variance was used for statistical analysis and p<0.05 was considered as significant.
resultsThe study design and patient exclusion criteria are summarised in figures 1 and 2, respectively. Patient infor-mation is summarised in table 1.
Cells from cancer tissue or pleural effusion (PE) samples obtained from each patient were limited; there-fore, the in vitro chemosensitivity tests were performed based on the clinical decision for treatment. The cryopre-served samples were thawed (viability routinely exceeded 90%; online supplementary S. figure 1), aggregates were made, then drug sensitivity tests were performed with drugs or drug combinations selected by the oncoteam (online supplementary S. figure 2). The in vitro viability
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from
Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505 3
Open access
Figure 2 Exclusion criteria. Out of the 120 patients only 14 met the inclusion criteria, 10 for solid tumours and 4 for pleural effusions. The exclusion criteria are named in the boxes along with the number of patients excluded from the study. RECIST, Response Evaluation Criteria In Solid Tumours.
test results were compared with the RECIST1.1 data.16 In vitro mean viability values at and below 0.8 (induction of cell death 0.2<) corresponded to patients with clinically SD, while a mean viability value of 0.9 and above (no or low level (<0.1) induction of cell death) corresponded to patients with clinically PD (figure 3A; online supple-mentary table 1). Cut- off values are explained in online supplementary S. figure 3. Sample numbers on the figure correspond to the patient numbers in table 1. The patient who donated the KRAS mutant solid tumour sample (S1) was PD during clinical observation, then following cisplat-in+pemetrexed combination therapy became stable (SD). S1 patient became PD again after changing the treatment to pemetrexed monotherapy and the RECIST result correlated with the in vitro chemosensitivity anal-ysis. The change in therapy was forced by severe adverse reactions to cisplatin. Patients S2 (wild type (WT)) and S9 (KRAS) responded well to cisplatin+vinorelbine combination as they were both SD at clinical examina-tion and in vitro testing. Patients S4 (KRAS), S7 (KRAS), S8 (undisclosed mutation status) and S10 (EGFR) were SD correlating to the in vitro analysis. Patient samples S3 (KRAS), S5 (KRAS) and S6 (KRAS) remained firmly non- responsive to therapy and clinically PD after evalua-tion (figure 3A). The in vitro test results correlated well with the clinical data (figure 3A). Among the PE samples, donor of PE sample 2 (PE2) was initially SD after cisplat-in+pemetrexed combination treatment but became PD when due to severe reactions to cisplatin, treatment was
changed to pementrexed monotherapy (figure 3A). The same chemosensitivity response was detected also in vitro. Discrepancies between clinical and in vitro evalu-ation were detected in some cases. Correcting the corre-sponding in vitro data with the time course of progression information (figure 3B), a stronger association between the in vitro viability analysis and patient response to therapy (figure 3C) was detected.
To investigate the possibility whether the above- mentioned in vitro drug sensitivity test could supplement the clinical decision- making process, a PE sample was selected for further studies. The patient who donated the sample did not respond to the clinically offered CBP–paclitaxel combination therapy (PD; figure 4). The in vitro chemosensitivity analysis using CBP–paclitaxel matched the clinical response (relative cell viability values were above 0.9, no induction of cell death; figure 4). Another, clinically approved combination for therapy in this particular case could have been CBP–pemetrexed. In vitro analysis of the sample using CBP–pemetrexed treat-ment of cell aggregates reduced cell viability below 0.8 (effective induction of cell death) that is in the SD range of the therapy response (figure 4).
dIscussIonAccording to the US Precision Medicine Initiative,21 the arsenal of precision medicine should be at the finger-tips of every oncologist. The clinical reality, however,
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from
4 Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505
Open access
Tab
le 1
C
linic
al d
ata
(Par
t A
con
tain
s d
ata
of p
atie
nts
don
atin
g so
lid t
umou
rs, w
hile
par
t B
con
tain
s a
list
of s
amp
les
obta
ined
from
ple
ural
effu
sion
s)
Sam
ple
nu
mb
erM
utat
ions
Dia
gno
sis
TN
MIn
terv
enti
on
Dat
e o
f sa
mp
ling
Clin
ical
the
rap
yIn
vit
roTy
pe
of
trea
tmen
tB
egin
ning
o
f th
erap
yE
nd o
f th
erap
y
Trea
tmen
t b
efo
re/a
fter
sa
mp
ling
RE
CIS
TR
EC
IST
d
ate
PD
- L1
A. P
atie
nt d
ata
of
solid
tum
our
s
Sol
id t
umor
s
1
KR
AS
NS
CLC
- A
den
occ.
pT1
bp
N1
Mx
Com
ple
te
tum
our
rese
ctio
n
27 O
ctob
er
2015
Ob
serv
atio
n–
27 O
ctob
er
2015
22 M
ay
2017
Aft
erP
D29
Mar
ch
2017
>50
%
(Pos
itive
)
pT1
bN
3M
1aC
isp
latin
+ p
emet
rexe
dTe
sted
Pal
liativ
e23
May
20
1709
Aug
ust
2017
Aft
erS
D25
Aug
ust
2017
T3N
3M
1cP
emet
rexe
d m
ono
Test
edP
allia
tive
17 O
ctob
er
2017
28
Nov
emb
er
2017
Aft
erP
D14
Dec
emb
er 2
017
2
WT
NS
CLC
- A
den
occ.
pT2
aN
1M
xC
omp
lete
tu
mou
r re
sect
ion
01
Dec
emb
er
2015
Cis
pla
tin +
vin
orel
bin
eTe
sted
Ad
juva
nt06
Jan
uary
20
1624
Jun
e 20
16A
fter
SD
–N
egat
ive
3
KR
AS
NS
CLC
- A
den
occ.
pT3
pN
2M
xC
omp
lete
tu
mou
r re
sect
ion
28 A
pril
20
16O
bse
rvat
ion
–29
Ap
ril
2016
29 J
une
2016
Aft
erP
D26
May
201
6N
egat
ive
pT3
pN
2M
1aC
isp
latin
+ p
emet
rexe
dTe
sted
Pal
liativ
e30
Jun
e 20
1630
Jun
e 20
16A
fter
PD
05 J
uly
2016
4
KR
AS
NS
CLC
- A
den
occ.
pT3
N0
M1b
Com
ple
te
tum
our
rese
ctio
n
03 M
ay
2016
Cis
pla
tin +
ge
mci
tab
ine
Test
edA
dju
vant
07 J
uly
2016
08 A
ugus
t 20
16A
fter
SD
06
Sep
tem
ber
20
17
Neg
ativ
e
5
KR
AS
NS
CLC
- A
den
occ.
pT3
aN
1M
xC
omp
lete
tu
mou
r re
sect
ion
26 J
une
2016
Cis
pla
tin +
vin
orel
bin
eTe
sted
Ad
juva
nt08
Aug
ust
2016
30
Sep
tem
ber
20
16
Aft
erS
D27
Ap
ril 2
017
Neg
ativ
e
6
KR
AS
NS
CLC
- A
den
occ.
pT2
ap
N2
M1c
Com
ple
te
tum
our
r ese
ctio
n
12
Sep
tem
ber
20
16
Car
bop
latin
+
gem
cita
bin
eTe
sted
Ad
juva
nt12
Oct
ober
20
1607
D
ecem
ber
20
16
Aft
erP
D24
Jan
uary
20
17<
1%
(Neg
ativ
e)
7
KR
AS
NS
CLC
-
Ad
eno
ccp
T2p
N1
Mx
Com
ple
te
tum
our
rese
ctio
n
05
Dec
emb
er
2016
Cis
pla
tin+
pem
etre
xed
Test
edA
dju
vant
21 F
ebru
ary
2017
03 M
ay
2017
Aft
erS
D23
May
201
7
8
NS
CLC
-
Ad
eno
ccp
T2a
pN
xM
xC
omp
lete
tu
mou
r re
sect
ion
05 J
anua
ry
2017
Car
bop
latin
–
pac
litax
elTe
sted
Ad
juva
nt28
Mar
ch
2017
15 J
une
2017
Aft
erS
D20
Jul
y 20
17
9
KR
AS
NS
CLC
-
Ad
eno
ccp
T2p
N1
Mx
Com
ple
te
tum
our
rese
ctio
n
14
Dec
emb
er
2015
Cis
pla
tin +
vin
orel
bin
eTe
sted
Ad
juva
nt18
Feb
ruar
y 20
1608
Ap
ril
2016
SD
08 J
uly
2016
10
EG
FRN
SC
LC -
A
den
o cc
pT2
ap
N2
Mx
Com
ple
te
tum
our
rese
ctio
n
19
Dec
emb
er
2016
Cis
pla
tin +
vin
orel
bin
eTe
sted
Ad
juva
nt09
Feb
ruar
y 20
1702
Mar
ch
2017
Aft
erS
D20
Mar
ch 2
017
pT2
ap
N2
Mx
Erlo
tinib
mon
oTe
sted
Pal
liativ
e18
Oct
ober
20
1713
Aug
ust
2018
Aft
erP
D13
Aug
ust
2018
Cont
inue
d
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from
Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505 5
Open access
Sam
ple
nu
mb
erM
utat
ions
Dia
gno
sis
TN
MIn
terv
enti
on
Dat
e o
f sa
mp
ling
Clin
ical
the
rap
yIn
vit
roTy
pe
of
trea
tmen
tB
egin
ning
o
f th
erap
yE
nd o
f th
erap
y
Trea
tmen
t b
efo
re/a
fter
sa
mp
ling
RE
CIS
TR
EC
IST
d
ate
PD
- L1
B. P
atie
nt d
ata
of
ple
ural
eff
usio
n d
ono
rs
Ple
ural
effu
sion
s
1
EG
FRN
SC
LC -
A
den
o cc
T3N
xM
1bTh
orac
ic
asp
iratio
n20
Feb
ruar
y 20
17G
efitin
ibTe
sted
Pal
liativ
e04
Feb
ruar
y 20
1514
Jun
e 20
17D
urin
gP
D14
Jun
e 20
17N
egat
ive
2
KR
AS
NS
CLC
-
Ad
eno
ccT2
aN
2M
1aTh
orac
ic
asp
iratio
n06
May
20
17C
isp
latin
+ p
emet
rexe
dTe
sted
Pal
liativ
e20
May
20
1628
Jul
y 20
17A
fter
SD
04 A
ugus
t 20
16N
egat
ive
T2a
N2
M1a
Pem
etre
xed
mon
oTe
sted
Pal
liativ
e19
S
epte
mb
er
2016
03
Nov
emb
er
2016
Aft
erP
D17
Nov
emb
er20
16
3
EG
FRN
SC
LC -
A
den
o cc
T4N
2M
1Th
orac
ic
asp
iratio
n25
Feb
ruar
y 20
16E
rlotin
ib m
ono
Test
edP
allia
tive
26 J
anua
ry
2016
28 J
uly
2016
Bef
ore
PD
13 J
uly
2017
Neg
ativ
e
T4N
2M
1O
sim
ertin
ib-
Pal
liativ
e31
Aug
ust
2016
30 J
une
2017
Aft
erP
D01
Aug
ust
2017
T4N
2M
1C
arb
opla
tin +
p
aclit
axel
Test
edP
allia
tive
09 A
ugus
t 20
1730
Aug
ust
2017
Aft
erP
D18
Sep
tem
ber
201
7
T4N
3M
1cP
emet
rexe
d m
ono
Test
edP
allia
tive
11 O
ctob
er
2017
05 J
anua
ry
2018
Aft
erP
D25
Jan
uary
201
8
4
WT
NS
CLC
-
Ad
eno
ccT2
aN
2M
1aTh
orac
ic
asp
iratio
n27
Jul
y 20
16C
arb
opla
tin +
p
aclit
axel
Test
edP
allia
tive
15 J
une
2016
26 A
ugus
t 20
16D
urin
gP
D08
S
epte
mb
er
2016
Neg
ativ
e
T4N
3M
1bE
rlotin
ib m
ono
Test
edP
allia
tive
04 O
ctob
er
2016
12
Dec
emb
er
2016
Aft
erP
D12
Dec
emb
er 2
016
The
info
rmat
ion
rang
es fr
om t
he a
ctua
l met
hod
of o
bta
inin
g th
e sa
mp
le, m
utat
ion
anal
ysis
, dia
gnos
is, s
tagi
ng (T
NM
=tu
mou
r, no
de,
met
asta
sis
stat
us) o
f the
dis
ease
and
ap
plie
d t
reat
men
t or
tre
atm
ents
and
clin
ical
re
spon
ses.
EG
FR, e
pid
erm
al g
row
th fa
ctor
rec
epto
r; K
RA
S, K
irste
n ra
t sa
rcom
a 2
vira
l onc
ogen
e ho
mol
og; N
SC
LC, n
on- s
mal
l cel
l lun
g ca
ncer
; PD
, pro
gres
sive
dis
ease
; PD
- L1,
pro
gram
med
dea
th li
gand
-1; R
EC
IST,
Res
pon
se
Eva
luat
ion
Crit
eria
In S
olid
Tum
ours
; SD
, sta
ble
dis
ease
.
Tab
le 1
C
ontin
ued
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from
6 Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505
Open access
Figure 3 Chemosensitivity analysis. (A) Three- dimensional (3D) aggregate cultures were treated with patient- specific chemotherapeutic agents as determined by the oncoteam. After incubation at 37°C for 48 hours in a 96- well plate, ATP levels corresponding to cell viability were determined using a 3D CellTiter Glo kit. In vitro viability data were compared with Response Evaluation Criteria In Solid Tumours (RECIST1.1) data when it became available. Patient- specific data are shown individually and marked with the patient identifying number used in the study. (B) Individual patient data shown in association of time laps to disease progression. (C) Percentage of correspondence between clinical RECIST1.1 information and in vitro analysis results. PD, progressive disease; SD, stable disease.
is different. Traditional chemotherapies are still the remaining treatment options for patients with stage III B or IV NSCLC tumours, that carry no targetable muta-tions, and are not positive for PD- L1. However, decision- making as chemosensitivity of the tumour is currently not tested routinely. Oncologists, who are bound by specific clinical guidelines, still need more than one treatment option to offer a patient. Even if the approved guidelines provide some choices, the fast progressing disease allows little time to select additional tests for the presence or absence of biomarkers indicating previously unpredicted therapeutic targets. The in vitro drug sensitivity analysis in the above study was performed to test whether the clin-ical effect of chemotherapeutic drug combinations could be tested using a simple and fast method where targeted therapy was not available for the patients. Compared with previous studies and test methods,22 we intentionally remained within the current routine clinical boundaries. Instead of using a vast number of mutation analyses23 and artificial intelligence24 to achieve better accuracy, we simply selected the patient population more carefully. For example, patients who were subjected to Avastin or immune checkpoint treatment were excluded from
the test, as the test tissue was lacking blood supply and contained no immune cells. Such strict selection criteria had the consequence of a drastically reduced number of patients whose in vitro and clinical data were suitable for comparison. Although the number of patients were limited in the study, the in vitro data indicate that in vitro chemosensitivity tests could aid clinical drug selection and potentially expand survival even for patients with advanced lung AC. The fact that we are able to test more than one chemotherapy combination in vitro (figure 4) raises the possibility that an approved in vitro drug sensi-tivity test could make clinical decision- making easier. The in vitro process is feasible and could be easily added to the decision- making process. Partly, because it does not require additional sample taking from the patient and does not increase the workload of clinical staff.
In conclusion, chemosensitivity tests could supplement the clinical diagnostic arsenal as: (1) the assays can be performed from a small number of cells (1000 cell/well); therefore, even samples from distinct metastatic sites can be tested if sample taking is clinically feasible. (2) Cryopreservation of tumour cells allows sufficient time to perform additional diagnostic tests. (3) The analysis
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from
Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505 7
Open access
Figure 4 Testing optional drug sensitivity. Clinical application of carboplatin–paclitaxel combination therapy resulted in progressive disease with a matching in vitro chemosensitivity analysis of cell viability values above 0.9. Treatment of tissue culture with carboplatin–pemetrexed in vitro reduced cell viability below 0.8 that is in the stable disease range of therapy response. Viability compared with the untreated control was significantly lower when cell cultures were incubated with carboplatin–pemetrexed combination (p<0.01). PE, pleural effusion; N.S., not significant.
provides information within 48 hours, which is vital for patients with fast progressing tumours.
Additionally, the above system could also be introduced into drug development. To reduce systemic toxicity, novel prodrug systems are being developed.25 Although the 3D tissue aggregate is not suitable to test most prodrugs, the toxic effects of the active metabolite can be tested in the above system.
The prediction of the clinical response to chemo-therapeutic drugs remains a major challenge in clinical oncology. If our simple and fast in vitro method were to be used to test chemosensitivity and if that test result is added to the patient’s full clinical assessment, a decision for therapy could be made with increased confidence.
Author affiliations1Internal Medicine, Pulmonology, The Medical School and Clinical Centre, University of Pecs, Pecs, Baranya, Hungary2Research, Humeltis Ltd, Pecs, Baranya, Hungary3Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Pecs, Pecs, Baranya, Hungary4Szentagothai Research Centre, University of Pecs, Pecs, Baranya, Hungary5Surgery, The Medical School and Clinical Centre, University of Pecs, Pecs, Baranya, Hungary6Pathology, The Medical School and Clinical Centre, University of Pecs, Pecs, Baranya, Hungary7Tumour Biology, National Korányi Institute of Pulmonology, Budapest, Hungary8Pulmonology, Semmelweis University, Budapest, Hungary
Acknowledgements The authors would like to thank Mary Keen, Emerita Professor of Pharmacology, University of Birmingham and Honorary Professor, University of Pecs for language editing.
contributors All authors contributed to the study design and data analysis, and reviewed and approved the manuscript.
Funding This work was supported in part by the University of Pecs KA Research Fund 2018 (to JEP); GINOP-2.3.2.-15-2016-00022, TUDFO/51757-1/2019- ITM and by Humeltis.
competing interests AS and JM- R: employees of Humeltis. JEP: received a grant and personal payments from Humeltis.
Patient consent for publication Not required.
ethics approval The project was approved by the Ethical Committee of the University of Pecs (2014- RIKEB-5329- EKK) and the Medical Research Council of Hungary (366/2015 (46945-1/2015/EKU).
Provenance and peer review Not commissioned; externally peer reviewed.
open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non- commercial. See: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
orcId idJudit E Pongracz http:// orcid. org/ 0000- 0002- 0278- 5556
RefeRenCes 1 Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2009. CA Cancer
J Clin 2009;59:225–49. 2 Langer CJ. Roles of EGFR and KRAS mutations in the treatment of
patients with non- small- cell lung cancer. P T 2011;36:263–79. 3 Pikor LA, Ramnarine VR, Lam S, et al. Genetic alterations defining
NSCLC subtypes and their therapeutic implications. Lung Cancer 2013;82:179–89.
4 Eberhardt WEE, De Ruysscher D, Weder W, et al. ESMO clinical practice guidelines: lung and chest tumours. Ann Oncol 2015;26:1573–88.
5 Planchard D, Popat S, Kerr K, et al. Metastatic non- small cell lung cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow- up. Ann Oncol 2018;29:iv192–237.
6 Leichsenring J, Horak P, Kreutzfeldt S, et al. Variant classification in precision oncology. Int J Cancer 2019;145:2996–3010.
7 Boot A, Huang MN, Ng AWT, et al. In- depth characterization of the cisplatin mutational signature in human cell lines and in esophageal and liver tumors. Genome Res 2018;28:654–65.
8 Daste A, Domblides C, Gross- Goupil M, et al. Immune checkpoint inhibitors and elderly people: A review. Eur J Cancer 2017;82:155–66.
9 Blom K, Nygren P, Larsson R, et al. Predictive value of ex vivo chemosensitivity assays for individualized cancer chemotherapy: a meta- analysis. SLAS Technol 2017;22:306–14.
10 Breslin S, O'Driscoll L. Three- dimensional cell culture: the missing link in drug discovery. Drug Discov Today 2013;18:240–9.
11 Deng X, Nakamura Y. Cancer precision medicine: from cancer screening to drug selection and personalized immunotherapy. Trends Pharmacol Sci 2017;38:15–24.
12 Dean EJ, Ward T, Pinilla C, et al. A small molecule inhibitor of XIAP induces apoptosis and synergises with vinorelbine and cisplatin in NSCLC. Br J Cancer 2010;102:97–103.
13 Wang S, Xie J, Li J, et al. Cisplatin suppresses the growth and proliferation of breast and cervical cancer cell lines by inhibiting integrin β5- mediated glycolysis. Am J Cancer Res 2016;6:1108–17.
14 Su WC, Chang SL, Chen TY, et al. Comparison of in vitro growth- inhibitory activity of carboplatin and cisplatin on leukemic cells and hematopoietic progenitors: the myelosuppressive activity of carboplatin may be greater than its antileukemic effect. Jpn J Clin Oncol 2000;30:562–7.
15 Biziota E, Briasoulis E, Mavroeidis L, et al. Cellular and molecular effects of metronomic vinorelbine and 4- O- deacetylvinorelbine on human umbilical vein endothelial cells. Anticancer Drugs 2016;27:216–24.
16 Wouters A, Pauwels B, Lardon F, et al. In vitro study on the schedule- dependency of the interaction between pemetrexed, gemcitabine and irradiation in non- small cell lung cancer and head and neck cancer cells. BMC Cancer 2010;10:441.
17 Zasadil LM, Andersen KA, Yeum D, et al. Cytotoxicity of paclitaxel in breast cancer is due to chromosome missegregation on multipolar spindles. Sci Transl Med 2014;6:229ra43.
18 Chin TM, Quinlan MP, Singh A, et al. Reduced erlotinib sensitivity of epidermal growth factor receptor- mutant non- small cell lung cancer following cisplatin exposure: a cell culture model of second- line erlotinib treatment. Clin Cancer Res 2008;14:6867–76.
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from
8 Papp E, et al. BMJ Open Resp Res 2020;7:e000505. doi:10.1136/bmjresp-2019-000505
Open access
19 Ono M, Hirata A, Kometani T, et al. Sensitivity to gefitinib (Iressa, ZD1839) in non- small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signal- regulated kinase 1/2 and EGF receptor/Akt pathway for proliferation. Mol Cancer Ther 2004;3:465–72.
20 Schwartz LH, Litière S, de Vries E, et al. RECIST 1.1- Update and clarification: from the RECIST Committee. Eur J Cancer 2016;62:132–7.
21 The White House. The precision medicine initiative: data- driven treatments as unique as your own body. Available: https:// obamawhitehouse. archives. gov/ blog/ 2015/ 01/ 30/ precision-
medicine- initiative- data- driven- treatments- unique- your- own- body [Accessed 5 Mar 2020].
22 Majumder B, Baraneedharan U, Thiyagarajan S, et al. Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity. Nat Commun 2015;6:1–14.
23 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74.
24 Bohannon J. Fears of an AI pioneer: Stuart Russell argues that AI is as dangerous as nuclear weapons. Science 2015;349:252.
25 Giang I, Boland EL, Poon GMK. Prodrug applications for targeted cancer therapy. AAPS J 2014;16:899–913.
copyright. on July 21, 2021 by guest. P
rotected byhttp://bm
jopenrespres.bmj.com
/B
MJ O
pen Resp R
es: first published as 10.1136/bmjresp-2019-000505 on 10 June 2020. D
ownloaded from